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DQ-Red BSA Trafficking Assay in Cultured Cells to Assess Cargo Delivery to Lysosomes
在培养细胞中评估物质运输至溶酶体的DQ-Red BSA转运分析法   

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Abstract

Lysosomes are the terminal end of the endocytic pathway having acidic environment required for active hydrolases that degrade the cargo delivered to these compartments. This process of cargo delivery and degradation by endo-lysosomes is a tightly regulated process and important for maintaining cellular homeostasis. Cargos like EGF (Epidermal Growth Factor), Dil-LDL (3,3’-Dioctadecylindocarbocyanine-Low Density Lipoprotein), Dextran, DQ-BSA (Dye Quenched-Bovine Serum Albumin) etc., are routinely used by researchers to analyze the role of various proteins in endocytic pathway. Trafficking of DQ-BSA in cells depleted of or over-expressing the gene of interest is a useful assay for identifying the role of various proteins in endocytic trafficking pathway. The protocol describes the DQ-Red BSA trafficking assay that can be used to study endocytic trafficking in various cell types.

Keywords: DQ-BSA(DQ-BSA), Lysosome(溶酶体), Cargo(物质), Endocytosis(胞吞作用), Cell culture(细胞培养), HeLa(HeLa)

Background

Cells are constantly exchanging materials with their extracellular environment and in this process they internalize cargo in vesicles at the plasma membrane. This internalized cargo is delivered to the early endosomes from where it either goes back to the plasma membrane via recycling endosomes or enters the canonical endocytic pathway. Once destined to be degraded, the cargo moves to late endosomes and finally fuses with lysosomes where the active hydrolases digest the cargo (Jovic et al., 2010). These endocytic compartments have characteristic pH of their lumen. The early endosomes have a pH in the range of 5.9-6.8, late-endosomes have pH range of 4.9-6.0, and lysosomes are most acidic with pH ranging from 4.5-5.0 (Maxfield and Yamashiro, 1987). The acidic environment of lysosomes is necessary for the activity of hydrolases present in its lumen and degradation of cargo (Garg et al., 2011; Khatter et al., 2015a and 2015b).

Here we discuss the trafficking assay using DQ-Red BSA as cargo, which is a BSA (bovine serum albumin) derived cargo heavily labeled with BODIPY TR-X dye, resulting in self quenching of the dye. Degradation of DQ-Red BSA in acidic, hydrolase active endo-lysosomes results in smaller protein fragments that have isolated fluorophores, hence de-quenching the dye that can be visualized as a bright fluorescence in cells. The excitation and emission maxima for this dye are ~590 nm and ~620 nm respectively. Trafficking of DQ-Red BSA can be used to study the delivery of cargo to lysosomes (Pols et al., 2013; Marwaha et al., 2017; see Figure 1). Under normal conditions, DQ-Red BSA traffics to lysosomes and is cleaved by lysosomal hydrolases, resulting in bright red fluorescent signal (Figure 2). Disruption of cargo delivery to lysosomes such as by depletion of a certain gene product or treatment with chemical inhibitors (such as Bafilomycin A) impairs proteolysis of DQ-Red BSA and thus weak or no fluorescence is observed (see Figure 3).


Figure 1. Schematic representation of DQ-Red BSA cargo uptake and processing in cells. DQ-Red BSA is endocytosed in cells and traffics through early endosomes to late endosomes which then fuse with acidic hydrolase containing lysosomes. This leads to the formation of endo-lysosomes that degrade DQ-Red BSA, de-quenching the fluorescence of the dye attached to this cargo. DQ-Red BSA is heavily labeled with fluorescent dyes that lead to quenching of their fluorescence (shown by pink fluorophores attached to the cargo). Once DQ-Red BSA has reached the degradative endo-lysosomes, the cargo is broken down into smaller fragments which lead to the de-quenching of the fluorophores (shown as bright red spots in endo-lysosomes).


Figure 2. De-quenching of DQ-Red BSA fluorescence at different time points post endocytosis. Representative single-plane confocal micrographs of HeLa cells showing fluorescence (red signal) of DQ-Red BSA after an uptake of 1 h (A) and 6 h (B) time points. At the end of the uptake time point, cells were fixed and stained with DAPI (blue) to mark the cell nucleus. A bright fluorescence punctae of DQ-Red BSA is visible post 6 h uptake as compared to 1 h, indicating its delivery to acidic compartments of the cell. Scale bars = 10 µm.


Figure 3. DQ-BSA uptake and delivery to lysosomes. Representative single-plane confocal micrographs of HeLa cells incubated with DQ-Red BSA in 1% serum containing media for indicated time point in the absence (A) or presence (B) of 100 nM Bafilomycin A1, a V-ATPase inhibitor that disrupts lysosome fusion. The cell nucleus is stained using DAPI (blue). Scale bars = 10 µm.

Materials and Reagents

  1. Coverglass slips (VWR, catalog number: 89015-725 )
  2. BD Falcon15 ml centrifuge tubes (Corning, Falcon®, catalog number: 352096 )
  3. BD Falcon 35 mm cell culture dish (Corning, Falcon®, catalog number: 353001 )
  4. 24-well plate (Corning, Falcon®, catalog number: 353226 )
  5. Glass slide (HiMedia Laboratories, catalog number: CG029 )
  6. HeLa cells (ATCC, catalog number: CCL-2 )
  7. siRNA (Dharmacon)
  8. Xtremegene HD transfection reagent (Roche)
  9. DMEM (Lonza, catalog number: 12-604F )
  10. NEAA (Thermo Fisher Scientific, GibcoTM, catalog number: 11140050 )
  11. GlutaMax (Thermo Fisher Scientific, GibcoTM, catalog number: 35050061 )
  12. HEPES (Thermo Fisher Scientific, GibcoTM, catalog number: 15630080 )
  13. DQ-Red BSA (Thermo Fisher Scientific, InvitrogenTM, catalog number: D12051 )
  14. DAPI (Thermo Fisher Scientific, InvitrogenTM, catalog number: D1306 )
  15. HI FBS (Thermo Fisher Scientific, GibcoTM, catalog number: 10082147 )
  16. 1x DPBS (Lonza, catalog number: 17-512F )
  17. 16% paraformaldehyde (PFA) (Electron Microscopy Sciences, catalog number: 15700 )
  18. Fluoromount G (Southern Biotech, catalog number: 0100-01 )
  19. Stock solution of DQ-Red BSA (see Recipes)
  20. DAPI stock solution (see Recipes)

Equipment

  1. CO2 incubator (Eppendorf, New BrunswickTM, model: Galaxy® 170 R )
  2. Confocal microscope (ZEISS, model: LSM 710 )

Software

  1. ImageJ software (NIH)

Procedure

  1. Trafficking–DQ-Red BSA in HeLa cells
    Note: This protocol provides general guidelines and a procedure to perform trafficking assay using DQ-Red BSA as a cargo in HeLa cells. For other cell types, check which culture media and culture supplements the cell line you are using requires before starting cultures.The suggested cell number, concentration of DQ-BSA to be used, and transfection conditions are provided as a starting point. We recommend users to optimize these conditions based on the cell types they are using for the assay in order to obtain best results.
    1. Cells are seeded on coverglass slips according to the experiment to be performed, for example siRNA transfection or plasmid transfection for over-expression studies.
      Note: For RNAi studies, seed 60,000 HeLa cells in a 35 mm cell culture dish containing 4 glass coverslips. After a period of 16 h, transfer individual glass coverslips to 24-well cell culture plate and siRNA treatment is performed as described in the next step. For overexpression related experiments, seed 0.18 million HeLa cells in a 35 mm cell culture dish containing 4 glass coverslips, and post 16 h of cell seeding, transfections is carried out as described below. The users are recommended to optimize the cell seeding density based on the cell type and their growth rates. In general, for RNAi studies that go upto 48-72 h, the starting cell density should be kept around 15-20%. For overexpression studies, the confluency of the cells should be around 40% at the day of transfection.
    2. Perform the gene silencing or overexpression of exogenous plasmids in cells. siRNA is transfected using Dharmafect (GE) reagent according to the manufacturer’s protocol and plasmid DNA transfection is carried out using XtremegeneHD (Roche).
    3. Proceed with the assay once effective gene silencing is achieved upon siRNA treatment or expression of an exogenous gene is observed upon transfection of plasmid in case of over-expression studies.
      Note: For RNAi studies using Dharmafect (GE) reagents, > 80% silencing of an endogenous gene can be achieved within 48 h after siRNA transfection into HeLa cells. Similarly, expression of exogenous plasmid can be achieved within 16 h post-transfection using Xtremegene HD transfection reagent (Roche) in HeLa cells. However, users are recommended to determine the optimal conditions (siRNA concentration, period of siRNA treatment, transfection reagent, concentration of plasmid etc.) in order to achieve effective silencing of gene of interest or expression of transfected gene for their cell line of choice. Once these parameters are optimized, proceed with the trafficking assay post transfection.
    4. Prepare the media for trafficking in a BD Falcon 15 ml centrifuge tube as follows:
      Trafficking media: DMEM + 1% serum + 1% NEAA + 1% GlutaMax + 1% HEPES
      Note: 300 µl for one well of a 24-well plate is sufficient to submerge the cells. Increase the volume of media required according to the dish size.
    5. Warm the media at 37 °C.
    6. DQ-BSA preparation: stock concentration–2 mg/ml; working concentration–10 µg/ml.
    7. Calculate the amount of DQ-Red BSA required and add to trafficking media pre-warmed at 37 °C. Keep this mix at 37 °C for another 5 min.
      Note: Always make sure to spin the stock DQ-Red BSA (see Recipes) tube prior to making the ligand-trafficking media mix i.e., DQ-Red BSA mixed with trafficking media. This will ensure settling down of any DQ-BSA aggregates that may interfere with the trafficking assay and analysis of results.
    8. Wash the cells seeded on coverslips once with pre-warmed 1x DPBS.
    9. Add the ligand-trafficking media mix to cells and incubate at 37 °C in a 5% CO2 humidified cell culture chamber for 1 h and 6 h, respectively.
    10. After the desired time points, fix cells with 4% PFA prepared in 1x DPBS for 10 min at room temperature.
    11. To mark the nucleus, DAPI staining can be performed. Make DAPI stock solution of 1 mg/ml in sterile water, and use 1:1,000 dilution for staining the cells. Incubate coverslips with DAPI containing solution for 20 min followed by three washes with 1x DPBS and mount the coverslips on a glass slide using Fluoromount G.
    12. For more information, readers are encouraged to see Video 1.

      Video 1. Demonstration of how to perform DQ-Red BSA trafficking assay. The video describes the steps of DQ-Red BSA trafficking assay including preparation of cargo containing media, important precautions to be taken and the method of performing the experiment.

  2. Imaging, quantification and data analysis
    1. The cells are imaged on a single plane using confocal microscope (LSM 710, ZEISS).
    2. Analysis the image using ImageJ software (NIH).
    3. Quantify the Corrected Total Cell Fluorescence (CTCF) as described:
      1. Open the desired confocal image (.lsm file format) in ImageJ software.
      2. Split channels and keep the window of desired channel open.
      3. Select the cell by making an outline using ‘freehand selection’ tool.
      4. Go to ‘Analyze’ → ‘Set measurements’ → ‘Select area, integrated density and mean gray value’.
      5. Go to ‘Analyze’ again → Click on ‘Measure’.
      6. A result window opens from where the values are copied to an Excel sheet.
      7. For measuring the background intensity of the image, select 5 random areas in the image that do not have cells and repeat the above mentioned steps.
      8. CTCF = Average Integrated intensity - (Average area x Average mean of background).
      9. Calculate the CTCF for each cell and plot the mean value. For more information, readers are encouraged to see Figure 4.


        Figure 4. Screenshots of important steps of CTCF analysis using ImageJ

    4. For analyzing the number of DQ-Red BSA puncta in cells, use ‘Analyze Particle’ tool of ImageJ software and perform the quantification as described below:
      1. Open the desired confocal image in ImageJ software.
      2. Split channels of the image and select the channel of your interest. Close rest of the channels.
      3. If there are multiple cells in an image, then crop out the cell of interest in the selected channel.
      4. Duplicate the image (Shortcut–Ctrl + Shift + D).
      5. Select any one of the two duplicate images and follow the steps written below.
      6. Go to ‘Image’ → ‘Adjust’ → ‘Threshold’ → select for the Algorithm from the drop box and see which one highlight the punctate structures best in your image or set the threshold ‘manually’ → ‘Apply’.
      7. Go to ‘Analyze’ → ‘Set measurements’ → select ‘Mean gray value, Area and Limit to threshold’.
      8. In the same window, redirect to–select the image other than that was selected in step 4e → Press ‘OK’.
      9. Go to ‘Analyze’ → ‘Analyze Particles’ → select ‘Display results and summarize’ → press ‘OK’.
      10. Two result windows will open. One will have the data from every individual particle whereas the other one will be the summary having the average of the values from all the particles in the cell analyzed. Copy the data to an Excel sheet and analyze.
      11. The result summary file shows the total count of particles analyzed, which is the number of DQ-Red BSA punctae in a cell. For more information, readers are encouraged to see Figure 5.


        Figure 5. Screenshots of important steps of particle count analysis using ImageJ

Data analysis

For calculating the ‘Corrected Total Cell Fluorescence (CTCF)’ and analyzing the ‘number of DQ-Red BSA punctae’, readers are encouraged to see Figures 4 and 5, respectively. For more information, please see ‘Marwaha, R., Arya, S. B., Jagga, D., Kaur, H., Tuli, A. and Sharma, M. (2017). The Rab7 effector PLEKHM1 binds Arl8b to promote cargo traffic to lysosomes. J Cell Biol 216(4): 1051-1070’.

Recipes

  1. Stock solution of DQ-Red BSA
    1. DQ-Red BSA is provided as 1 mg lyophilized powder
    2. To make a stock of 2 mg/ml of DQ-Red BSA, dissolve in 500 µl 1x DPBS
    3. Mix thoroughly by vortexing and pipetting
    4. Store at 4 °C after reconstitution
    5. Protect from light by covering the vial with an aluminum foil
  2. DAPI stock solution
    1 mg/ml in sterile water
    Note: Use stock solution at a 1:1,000 dilution for staining cells

Acknowledgments

This protocol was adapted and modified from a previously published study (Pols et al., 2013). The authors would like to thank Imran Sayeed (third year BS-MS student at IISER Mohali) for his contribution in preparing the video. R. Marwaha acknowledges financial support from the Indian Institute of Science Education and Research Mohali (IISER Mohali). M. Sharma acknowledges financial support from the Wellcome Trust/Department of Biotechnology (DBT). This work was supported by the Wellcome Trust/DBT India Alliance Intermediate Fellowship awarded to M. Sharma (IA/I/12/1/500523). M. Sharma also acknowledges the infrastructure and financial support from IISER Mohali. The authors declare no competing financial interests.

References

  1. Garg, S., Sharma, M., Ung, C., Tuli, A., Barral, D. C., Hava, D. L., Veerapen, N., Besra, G. S., Hacohen, N. and Brenner, M. B. (2011). Lysosomal trafficking, antigen presentation, and microbial killing are controlled by the Arf-like GTPase Arl8b. Immunity 35(2): 182-193.
  2. Jovic, M., Sharma, M., Rahajeng, J. and Caplan, S. (2010). The early endosome: a busy sorting station for proteins at the crossroads. Histol Histopathol 25(1): 99-112.
  3. Khatter, D., Raina, V. B., Dwivedi, D., Sindhwani, A., Bahl, S. and Sharma, M. (2015b). The small GTPase Arl8b regulates assembly of the mammalian HOPS complex on lysosomes. J Cell Sci 128(9): 1746-1761.
  4. Khatter, D., Sindhwani, A. and Sharma, M. (2015a). Arf-like GTPase Arl8: Moving from the periphery to the center of lysosomal biology. Cell Logist 5(3): e1086501.
  5. Marwaha, R., Arya, S. B., Jagga, D., Kaur, H., Tuli, A. and Sharma, M. (2017). The Rab7 effector PLEKHM1 binds Arl8b to promote cargo traffic to lysosomes. J Cell Biol 216(4): 1051-1070.
  6. Maxfield F. R. and Yamashiro D.J. (1987). Endosome acidification and the pathways of receptor-mediated endocytosis. Adv Exp Med Biol 225:189-98.
  7. Pols, M. S., ten Brink, C., Gosavi, P., Oorschot, V. and Klumperman, J. (2013). The HOPS proteins hVps41 and hVps39 are required for homotypic and heterotypic late endosome fusion. Traffic 14(2): 219-232.

简介

溶酶体是具有降解递送到这些隔室的货物的活性水解酶所需的酸性环境的内吞途径的末端。 这种内溶溶酶体的货物递送和降解过程是一个严格调节的过程,对于维持细胞体内平衡是重要的。 Cargos如EGF(表皮生长因子),Dil-LDL(3,3'-二十八碳基碳代花青 - 低密度脂蛋白),葡聚糖,DQ-BSA(染料淬灭 - 牛血清白蛋白)等常规 由研究人员用来分析各种蛋白质在内吞途径中的作用。 在缺乏或过表达感兴趣的基因的细胞中的DQ-BSA的贩运是用于鉴定各种蛋白质在内吞运输途径中的作用的有用测定。 该方案描述了可用于研究各种细胞类型的吞噬运输的DQ-Red BSA运输测定。
【背景】细胞不断地与其细胞外环境交换物质,并且在这个过程中,它们将货物内部化在质膜的囊泡中。这种内部货物被运送到早期的内体,从那里它可以通过再循环内体回到质膜或进入规范的内吞途径。一旦被注定要退化,货物就会移动到后期的内体,并最终与溶酶体融合,其中活性水解酶消化货物(Jovic等人,2010)。这些内吞室具有其内腔的特征pH。早期内体体内的pH值范围为5.9-6.8,晚期内体的pH范围为4.9-6.0,溶酶体最为酸性,pH范围为4.5-5.0(Maxfield和Yamashiro,1987)。溶酶体的酸性环境对于存在于其管腔中的水解酶和货物降解的活性是必需的(Garg等人,2011; Khatter等人,2015a和2015b )。
在这里,我们讨论使用DQ-Red BSA作为货物的贩运测定,货物是用BODIPY TR-X染料重度标记的BSA(牛血清白蛋白)衍生货物,导致染料自熄。在酸性水解酶活性溶酶体中,DQ-红色BSA的降解导致较小的具有分离的荧光团的蛋白质片段,从而使染色剂熄灭,可以将其显现为细胞中的明亮荧光。该染料的激发和发射最大值分别为〜590nm和〜620nm。 DQ-Red BSA的贩运可以用于研究货物运送到溶酶体(Pols等人,2013; Marwaha等人,2017;参见图1) 。在正常条件下,DQ-Red BSA流向溶酶体,并被溶酶体水解酶切割,导致明亮的红色荧光信号(图2)。通过消耗某些基因产物或用化学抑制剂(如Bafilomycin A处理)将溶剂体的运送中断破坏DQ-Red BSA的蛋白水解,因此观察到弱或无荧光(见图3)。


图1. DQ-Red BSA载体在细胞中的吸收和加工的方法表征。DQ-Red BSA在细胞内吞,通过早期内体进入后期内体,然后与含有溶酶体的酸性水解酶融合。这导致形成降解DQ-Red BSA的内溶酶体,消除附着在该货物上的染料的荧光。 DQ-Red BSA被荧光染料严重标记,导致荧光猝灭(由附着在货物上的粉红色荧光团显示)。一旦DQ-Red BSA已经达到降解内溶酶体,则货物被分解成较小的片段,这导致荧光团的去猝灭(在内溶素体中显示为亮红色斑点)。


图2.去内吞后不同时间点DQ-Red BSA荧光的去猝灭代表性的单层共聚焦显微照片,显示DQ-Red BSA吸收后的荧光(红色信号) 1 h(A)和6 h(B)时间点。在摄取时间结束时,将细胞固定并用DAPI(蓝色)染色以标记细胞核。与1小时相比,6小时摄取后DQ-Red BSA的明亮的荧光斑点可见,表明其递送到细胞的酸性隔室。比例尺=10μm。

材料和试剂

  1. 盖玻片(VWR,目录号:89015-725)
  2. BD Falcon15ml离心管(Corning,Falcon ®,目录号:352096)
  3. BD Falcon 35 mm细胞培养皿(Corning,Falcon ®,目录号:353001)
  4. 24孔板(Corning,Falcon ®,目录号:353226)
  5. 玻璃滑梯(HiMedia Laboratories,目录号:CG029)
  6. HeLa细胞(ATCC,目录号:CCL-2)
  7. siRNA(Dharmacon)
  8. Xtremegene HD转染试剂(Roche)
  9. DMEM(Lonza,目录号:12-604F)
  10. NEAA(Thermo Fisher Scientific,Gibco TM ,目录号:11140050)
  11. GlutaMax(Thermo Fisher Scientific,Gibco TM ,目录号:35050061)
  12. HEPES(Thermo Fisher Scientific,Gibco TM ,目录号:15630080)
  13. DQ-Red BSA(Thermo Fisher Scientific,Invitrogen TM,目录号:D12051)
  14. DAPI(Thermo Fisher Scientific,Invitrogen TM,目录号:D1306)
  15. HI FBS(Thermo Fisher Scientific,Gibco TM ,目录号:10082147)
  16. 1x DPBS(Lonza,目录号:17-512F)
  17. 16%多聚甲醛(PFA)(电子显微镜科学,目录号:15700)
  18. Fluoromount G(Southern Biotech,目录号:0100-01)
  19. DQ-Red BSA的储备溶液(参见食谱)
  20. DAPI库存解决方案(见配方)

设备

  1. CO 2培养箱(Eppendorf,New Brunswick TM,型号:Galaxy 170R)
  2. 共焦显微镜(ZEISS,型号:LSM 710)

软件

  1. ImageJ软件(NIH)

程序

  1. HeLa细胞中的贩运DQ-红色BSA
    注意:本协议提供了使用DQ-Red BSA作为HeLa细胞中的货物进行贩运测定的一般准则和程序。对于其他细胞类型,检查哪些培养基和培养基补充您正在使用的细胞系,然后开始培养。提供的细胞数量,使用的DQ-BSA的浓度和转染条件作为起点。我们建议用户根据用于测定的细胞类型优化这些条件,以获得最佳效果
    1. 根据要进行的实验,将细胞接种在盖玻片上,例如siRNA转染或用于过表达研究的质粒转染。
      注意:对于RNAi研究,在含有4个玻璃盖玻片的35mm细胞培养皿中种植60,000个HeLa细胞。 16小时后,将单独的玻璃盖玻片转移到24孔细胞培养板上,如下一步所述进行siRNA处理。对于过表达相关实验,在含有4个玻璃盖玻片的35mm细胞培养皿中培养0.18Mg HeLa细胞,并在细胞接种16小时后进行转染,如下所述进行。推荐用户根据细胞类型及其生长速度优化细胞接种密度。一般来说,对于高达48-72小时的RNAi研究,起始细胞密度应保持在15-20%左右。对于过表达研究,转染当天细胞的汇合度应为约40%。
    2. 在细胞中进行外源质粒的基因沉默或过表达。使用Dharmafect(GE)试剂根据制造商的方案转染siRNA,并使用XtremegeneHD(Roche)进行质粒DNA转染。
    3. 一旦siRNA处理实现了有效的基因沉默,则进行测定,或者在过表达研究的情况下转染质粒时观察到外源基因的表达。
      注意:对于使用Dharmafect(GE)试剂的RNAi研究,在siRNA转染HeLa细胞后48小时内可以实现内源性基因的80%沉默。类似地,在HeLa细胞中使用Xtremegene HD转染试剂(Roche)可以在转染后16小时内实现外源质粒的表达。然而,建议用户确定最佳条件(siRNA浓度,siRNA处理时间,转染试剂,质粒浓度等),以实现其所选择的细胞系的感兴趣基因或转染基因表达的有效沉默。一旦优化了这些参数,请继续进行转染后的检测。
    4. 准备媒体贩运BD Falcon 15 ml离心管,如下所示:
      贩运媒介:DMEM + 1%血清+ 1%NEAA + 1%GlutaMax + 1%HEPES
      注意:一个24孔板的一个孔300μl就足以淹没细胞。增加所需介质的数量,根据菜的大小。
    5. 在37°C温暖媒体。
    6. DQ-BSA制备:储备浓度-2mg / ml;工作浓度为10μg/ ml
    7. 计算所需的DQ-Red BSA的量,并添加到在37°C预热的贩卖媒体。将此混合物在37°C再保持5分钟。
      注意:始终确保在制作配体运输介质混合物即DQ-Red BSA与贩运介质混合之前旋转库存DQ-Red BSA(参见食谱)管。 此将确保可能干扰贩运测定和结果分析的任何DQ-BSA聚集体。
    8. 用预热的1x DPBS将接种在盖玻片上的细胞洗涤一次。
    9. 将配体运输培养基混合物添加到细胞中,并在37℃下在5%CO 2加湿细胞培养室中孵育1小时和6小时。
    10. 在所需的时间点后,在室温下将在1x DPBS中制备的4%PFA的细胞固定10分钟
    11. 为了标记细胞核,可以进行DAPI染色。在无菌水中制备1mg / ml的DAPI储备溶液,并用1:1,000稀释液染色细胞。用含有DAPI的溶液孵育盖玻片20分钟,然后用1x DPBS洗涤3次,并使用Fluoromount G将玻片盖上玻片。
    12. 有关更多信息,请鼓励读者查看视频1.

      Video 1. Demonstration of how to perform DQ-Red BSA trafficking assay. The video describes the steps of DQ-Red BSA trafficking assay including preparation of cargo containing media, important precautions to be taken and the method of performing the experiment.

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player


  2. 成像,量化和数据分析
    1. 使用共聚焦显微镜(LSM 710,ZEISS)在单个平面上成像细胞。
    2. 使用ImageJ软件(NIH)分析图像。
    3. 量化校正的总细胞荧光(CTCF),如下所述:
      1. 在ImageJ软件中打开所需的共焦图像(.lsm文件格式)。
      2. 拆分通道并保持所需通道的窗口打开。
      3. 使用"手写选择"工具制作轮廓来选择单元格。
      4. 转到"分析"→"设置测量"→"选择区域,集成密度和平均灰度值"
      5. 再次转到"分析"→点击"测量"。
      6. 打开结果窗口,将值复制到Excel工作表。
      7. 为了测量图像的背景强度,请选择图像中没有单元格的5个随机区域,并重复上述步骤。
      8. CTCF =平均综合强度 - (平均面积×平均背景平均值)
      9. 计算每个单元格的CTCF并绘制平均值。有关更多信息,请鼓励读者查看图4.


        图4.使用ImageJ的CTCF分析的重要步骤截图

    4. 为了分析单元格中的DQ-Red BSA点数,使用ImageJ软件的"Analyze Particle"工具进行如下定量:
      1. 在ImageJ软件中打开所需的共焦图像。
      2. 拆分图像的通道,并选择您感兴趣的频道。关闭其余频道。
      3. 如果图像中有多个单元格,则将所选通道中感兴趣的单元格裁出。
      4. 复制图像(快捷键Ctrl + Shift + D)。
      5. 选择两个重复图像中的任何一个,并按照以下步骤执行。
      6. 转到"图像"→"调整"→"阈值"→从下拉框中选择算法,看看哪一个突出显示图像中的点状结构,或手动设置阈值→"应用"。
      7. 转到"分析"→"设置测量"→选择"平均灰度值,面积和限制到阈值"
      8. 在同一窗口中,重定向至选择第4步中选择的图像→按"确定"。
      9. 转到"分析"→"分析粒子"→选择"显示结果并进行总结"→按"确定"。
      10. 将打开两个结果窗口。人们将具有来自每个粒子的数据,而另一个将是具有所分析的细胞中所有粒子的平均值的总结。将数据复制到Excel表并分析。
      11. 结果汇总文件显示分析的颗粒总数,这是一个单元格中DQ-Red BSA点的数量。有关更多信息,请鼓励读者查看图5.


        图5.使用ImageJ进行粒子计数分析的重要步骤的截图

数据分析

为了计算"校正总细胞荧光(CTCF)"并分析"DQ-Red BSA punctae"的数量,鼓励读者分别参见图4和图5。有关更多信息,请参见"Marwaha,R.,Arya,S.B.Jagga,D.,Kaur,H.,Tuli,A.和Sharma,M。(2017)。 Rab7效应物PLEKHM1结合Arl8b以促进对溶酶体的货运。 J Cell Biol 216(4):1051-1070'。

食谱

  1. DQ-Red BSA的储备溶液
    1. DQ-Red BSA作为1mg冻干粉末提供
    2. 制备2mg / ml DQ-Red BSA的原液,溶于500μl1x DPBS中
    3. 通过旋涡和移液彻底混合
    4. 重建后在4°C储存
    5. 用铝箔覆盖小瓶避免光照
  2. DAPI库存解决方案
    1毫克/毫升无菌水
    注意:使用1:1,000稀释液的染色溶液染色细胞

致谢

该方案已经从先前发表的研究(Pols等人,2013)进行了修改和修改。作者要感谢Imran Sayeed(在伊斯坦布尔莫哈利举行的第三年的BS-MS学生)为准备录像带做出了贡献。 R. Marwaha承认印度科学教育与研究所Mohali(IISER Mohali)的财政支持。谢尔玛(Sharma)承认了惠康信托基金/生物科技部(DBT)的财务支持。这项工作得到了授予M.Sharma(IA / I / 12/1/500523)的维康信托基金/ DBT印度联盟中级奖学金的支持。夏尔马还承认了伊斯兰堡的Mohali的基础设施和财政支持。作者宣称没有竞争的经济利益。

参考

  1. Garg,S.,Sharma,M.,Ung,C.,Tuli,A.,Barral,D.C.,Hava,D.L.,Veerapen,N.,Besra,G.S.,Hacohen,N。和Brenner,M.B。(2011)。 溶酶体贩运,抗原呈递和微生物杀伤由Arf样GTPase Arl8b控制。 a> 免疫 35(2):182-193。
  2. Jovic,M.,Sharma,M.,Rahajeng,J.and Caplan,S。(2010)。 早期内体:在十字路口的蛋白质繁忙的分拣站。 Histol Histopathol 25(1):99-112。
  3. Khatter,D.,Raina,V. B.,Dwivedi,D.,Sindhwani,A.,Bahl,S。和Sharma,M。(2015b)。 小型GTPase Arl8b调节哺乳动物HOPS复合物在溶酶体上的组装。 J Cell Sci 128(9):1746-1761。
  4. Khatter,D.,Sindhwani,A.和Sharma,M.(2015a)。 类似Arf的GTPase Arl8:从外围移动到溶酶体生物学的中心。 Cell Logist 5(3):e1086501。
  5. Marwaha,R.,Arya,S.B.,Jagga,D.,Kaur,H.,Tuli,A.和Sharma,M。(2017)。 Rab7效应器PLEKHM1结合Arl8b以促进货运流向溶酶体。 Cell Biol 216(4):1051-1070。
  6. Maxfield F. R.和Yamashiro D.J. (1987年)。 内质子酸化和受体介导的内吞作用的途径。 Adv Exp Med Biol 225:189-98。
  7. Pols,M. S.,ten Brink,C.,Gosavi,P.,Oorschot,V.and Klumperman,J。(2013)。 HOPS蛋白hVps41和hVps39是同型和异型晚期内体融合所必需的。 Traffic 14(2):219-232。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Marwaha, R. and Sharma, M. (2017). DQ-Red BSA Trafficking Assay in Cultured Cells to Assess Cargo Delivery to Lysosomes. Bio-protocol 7(19): e2571. DOI: 10.21769/BioProtoc.2571.
  2. Marwaha, R., Arya, S. B., Jagga, D., Kaur, H., Tuli, A. and Sharma, M. (2017). The Rab7 effector PLEKHM1 binds Arl8b to promote cargo traffic to lysosomes. J Cell Biol 216(4): 1051-1070.
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